Project Summary/Abstract The long-term goal of these investigations is to develop methods based on high- throughput DNA sequencing for determining neuronal circuitry. Neurons transmit information to distant brain regions via long-range axonal projections. In some cases, functionally distinct populations of neurons are intermingled within a small region. Disruptions of connectivity may underlie many neuropsychiatric disorders including autism and schizophrenia. At present, neuroanatomical techniques?particularly those with single neuron resolution?are expensive and labor intensive. We have recently introduced a new technique, MAPseq, for the high-throughput mapping of projections. The core idea underlying MAPseq is to tag each neuron with many copies of a unique RNA barcode. Barcodes consisting of 30 random nucleotides have sufficient diversity to uniquely label every neuron in the mouse neocortex with high probability. The key advantage of barcoding is that it eliminates the need to trace neuronal processes across sections of tissue. Because the barcode is present throughout the neuron, the association between a distant neurite and its parent process is readily established without the need for reconstructing intervening sections. In this way, even long-range neuronal projections and connections can be determined. By transforming circuit mapping into a problem of DNA sequencing, we leverage the remarkable advances in next-generation DNA sequencing. The central goal of this proposal is to extend MAPseq to increase throughput and spatial resolution. The development of a high-throughput method for determining neuronal projections will have important implications for understanding normal neuronal circuitry, and how this circuitry is disrupted in animal models of neuropsychiatric disorders like autism and schizophrenia.